Bovine mammary glands were inoculated intracisternally with a streptomycin-resistant (SR) strain of Corynebacterium bovis to determine the number of colony-forming units (CFU) required to induce colonization and to maintain persistence of C bovis colonization throughout lactation and involution. Streptomycin resistance was used as a strain marker. Uninfected quarters in cows during midlactation were challenge exposed with successively higher numbers of SR C bovis until all quarters became colonized. Inoculum containing 790 CFU of SR C bovis established colonization in only 7 of 38 quarters. Colonization persisted in only 4 of these quarters by 23 days after inoculation. Eleven quarters were reinoculated with higher numbers of SR C bovis, and all became colonized by the time challenge-exposure inoculum contained 8 X 10(4) CFU. Colonization persisted throughout the 93-day experimental period. Somatic cell counts were significantly (P less than 0.01) higher in SR C bovis-colonized quarters after inoculation than before. Sixteen additional quarters were inoculated with a mean number of 8 X 10(4) CFU of SR C bovis 7 days before suppression of lactation. All quarters became colonized, and SR C bovis was shed during the experimental period; throughout the nonlactating and peripartum periods, high numbers of SR C bovis in pure culture were shed from 13 of 16 quarters.